The development of hybrid plant breeding has allowed for considerable advances in quality and quantity of crops that are produced. Increased yield and the combination of desirable characteristics, such as resistance to disease and insects, heat and drought tolerance, and variations in plant composition are all possible, in part, due to hybridization procedures. Hybridization procedures rely on the contribution of pollen from a male parent plant to a female parent plant in order to produce resulting hybrids.
Plants may self-pollinate if pollen from one flower is transferred to the same or a different flower of the same plant. Alternatively, plants may cross-pollinate if the pollen originates in a flower from a different plant. Maize plants (Zea mays) may be bred using both self-pollination and cross-pollination techniques. Maize plants have male flowers, which are located on the tassel, and female flowers, which are located on the ear of the same plant. Natural pollination in maize occurs when pollen from the tassels reaches the silks that are found at the tops of the incipient ears. Importantly, the development of maize hybrids relies upon male sterility systems.
The development of maize hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the resultant crosses. Pedigree breeding and recurrent selection are two breeding methods that may be used to develop inbred lines from maize populations. Breeding programs combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. A hybrid maize variety is the cross of two such inbred lines, each of which may have one or more desirable characteristics absent in one, or complementing the other. The new inbred plants are crossed with other inbred lines and the resultant hybrids from these crosses are evaluated to determine which are more desirable. The hybrid progeny from the first generation are designated F1. In the development of hybrids, only the F1 hybrids are sought. The F1 hybrid is typically more vigorous than its inbred parents. This hybrid vigor, termed “heterosis,” typically leads to more desirable traits, for example, increased vegetative growth and increased yield.
Hybrid maize seed can be produced by a male sterility system incorporating manual detasseling. To produce hybrid seed, the tassel is removed from the growing female inbred parent, which can be proximately planted in various alternating row patterns with the male inbred parent. Consequently, provided that there is sufficient isolation from foreign maize pollen, the ears of the female inbred will be fertilized only with pollen from the male inbred. The resulting seed is termed hybrid F1 seed.
However, manual detasseling is labor-intensive and costly. Manual detasseling is also often ineffective because in some instances environmental variation in plant development can result in plants tasseling after manual detasseling of the female parent plant is completed or because a detasseler might not completely remove the tassel of a female inbred plant. If detasseling is ineffective, the female plant will successfully shed pollen and some female plants will be self-pollinated. This will result in seed of the female inbred being undesirably harvested along with the hybrid seed which is normally produced. Female inbred seed is not as productive as F1 seed. In addition, the presence of female inbred seed can represent a germplasm security risk for the producer of the hybrid seed.
A female inbred plant can also be mechanically detasseled by a machine. Mechanical detasseling is approximately as reliable as hand detasseling, but is faster and less expensive. However, most detasseling machines produces more damage to the plants than hand detasseling. Thus, neither manual nor mechanical detasseling is entirely satisfactory at the present time.
Genetic male sterility is an alternative method that may be advantageously used in hybrid seed production. The laborious detasseling process can desirably be avoided in some genotypes by using cytoplasmic male-sterile inbred plants. In the absence of a fertility restorer gene, plants of a cytoplasmic male-sterile inbred are male sterile as a result of factors resulting from the cytoplasmic, as opposed to the nuclear, genome. Therefore, the characteristic of male sterility is inherited exclusively through the female parent in maize plants, since only the female provides cytoplasm to the fertilized seed. Cytoplasmic male-sterile plants are fertilized with pollen from another inbred plant that is not male-sterile. Pollen from the second inbred plant may or may not contribute genes that make the hybrid plants male-fertile. Typically, seed from detasseled normal maize and cytoplasmic male-sterile-produced seed of the same hybrid must be blended to ensure that adequate pollen loads are available for fertilization when the hybrid plants are grown and to ensure cytoplasmic diversity.
Drawbacks to use of cytoplasmic male sterility (CMS) as a system for the production of hybrid seed include the association of specific variants to CMS with susceptibility to certain crop diseases. See, e.g., Beckett (1971) Crop Science 11:724-6. This problem has specifically discouraged the use of the CMS-T variant in the production of hybrid maize seed, and has had a negative impact on the use of CMS in maize in general.
Cytoplasmic male sterility is the maternally inherited inability to produce functional pollen. More than 40 sources of CMS have been found and classified into three major groups by differential fertility restoration reactions. These groups are designated as CMS-T (Texas), CMS-S (USDA), and CMS-S (Charrua) (Beckett, 1971). In the CMS-T group, two dominant genes, Rf1 and Rf2, which are located on chromosomes 3 and 9, respectively, are required for the restoration of pollen fertility (Duvick, 1965). The S-cytoplasm is restored by a single gene, Rf3, which has been mapped on chromosome 2 (Laughnan and Gabay, 1978).
In maize, the restorer of the S type of CMS behaves as a gametophytic trait. Maize plants with S cytoplasm are restored by the single dominant gene, Rf3, which was mapped to the long arm of chromosome 2 and located between the whp1 and bnl17.14 loci (Kamps and Chase, 1997). Tie (2006) reported that Rf3 was associated with SSR markers umc1525 and bn1g1520 at distances of 2.3 and 8.9 cM, respectively. Zhang et al. (2006) identified three amplified fragment length polymorphism markers that were tightly linked to the Rf3 gene.
Heterozygous (Rf3/rf3) CMS-S plants are semi-fertile, shedding approximately 50% abortive collapsed pollen containing the rf3 allele and 50% starch-filled fertile pollen containing the Rf3 allele. The rf3 allele in Rf3/rf3 plants cannot be transferred to progeny through sterile pollen, thus generating sterile plants in F2 generation (Tie et al., 2006). This type of inheritance makes it very difficult to collect accurate phenotypic data from an F2 mapping population. Traditional methods for identifying mutations are labor and time-intensive, whole-genome sequencing was considered as an approach to determine the differences between CMS-S and restorer lines. At the same time, a backcross 1 (BC1) mapping population was designed to evaluate the identified mutations. A BC1 mapping population is advantageously more useful to evaluate phenotypes. Individuals from a backcross population have either Rf3/rf3 or rf3/rf3 genotypes, and thus there is no need to distinguish fully fertile phenotype from partially fertile phenotype during the phenotyping process.
Molecular markers are particularly useful for accelerating the process of introducing a gene or quantitative trait loci (QTL) into an elite cultivar or breeding line via backcrossing. Markers linked to the gene can be used to select plants possessing the desired trait, and markers throughout the genome can be used to select plants that are genetically similar to the recurrent parent (Young and Tanksley (1989) Theor. Appl. Genet. 77:95-101; Hospital et al. (1992) Genetics 132:1199-210).
Most of the plant fertility restorer genes have been cloned via a map-based cloning strategy. To date, five restorer genes have been isolated from several plant species including maize (Zea Mays L.) (Cui et al. (1996) Science 272:1334-6; Liu et al. (2001) Plant Cell 13:1063-78), petunia (Petunia hybrida) (Bentolila et al. (2002) Proc. Natl. Acad. Sci. USA 99:10887-92, radish (Raphanus sativus L.) (Brown et al. (2003) Plant J. 35:262-72; Desloire et al. (2003) EMBO Rep. 4:1-7; Koizuka et al. (2003) Plant J. 34:407-15), sorghum (Sorghum bicolor L.) (Klein et al. (2005) Theor. Appl. Genet. 111:994-1012) and rice (Oryza sativa L.) (Kazama and Toriyama (2003) FEBS Lett. 544:99-102; Akagi et al. (2004) Theor. Appl. Genet. 108:1449-57; Komori et al. (2004) Plant J. 37:315-25; Wang et al. (2006) Plant Cell 18:676-87. All of the identified restorer genes, except for Rf2 in maize, encode different pentatricopeptide repeat (PPR) proteins. The PPR protein contains 2 to 27 repeats of 35 amino acids, referred to as PPR motifs (Small and Peeters, 2000). Many PPR proteins are targeted to mitochondria where the CMS-associated genes and products are located (Lurin et al., 2004).
Additional information regarding fertility restorer genes from maize, rice, petunia, and radish may be found in U.S. Patent Application Ser. No. US2006/0253931, and in U.S. Pat. Nos. 5,981,833; 5,624,842; 4,569,152; 6,951,970; 6,392,127; 7,612,251; 7,314,971; 7,017,375; 7,164,058; and 5,644,066, all of which are incorporated herein by reference.